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Abstract:

Alternative treatments are provided for use in the inhibition, decrease,
therapeutic or prophylactic support of a Bdellovibrio and like organism
(BALO) Gram negative prey bacterial infection in a host.

Claims:

1. A method to decrease, treat or provide prophylactic support to a
Bdellovibrio and like organism (BALO) Gram negative prey bacterial
infection in a host, said method comprising administering a BALO and a
bacteriophage combination to a prey bacterial infection in a ratio of
from about ten to 1 to about 1 to one hundred, to inhibit, decrease,
treat, or provide prophylactic support against the growth of a BALO Gram
negative prey bacteria infected host, and wherein the BALO and
bacteriophage combination are employed in a single combined preparation,
and further wherein the Gram negative prey bacteria decreases by from
about 1 log to from about 5 logs.

5. The method of claim 1, wherein said bacterial infection is an
infection comprising a skin burn, skin infection, skin wound, a lung
infection, an ocular infection, an ear infection, a sinus infection, a
respiratory infection, a urinary tract infection, a infection from a
medical device, an infection from a medical equipment or an infection
from an implant.

6. The method of claim 5, wherein said infection is an ear infection.

7. The method of claim 5, wherein said infection is a urinary tract
infection.

8. The method of claim 5, wherein said infection is a skin infection.

9. The method of claim 5, wherein said infection is a skin wound
infection.

10. The method of claim 5, wherein said infection is a respiratory
infection.

11. The method of claim 5, wherein said infection is a sinus infection.

12. The method of claim 5, wherein said infection is an ocular infection.

13. The method of claim 5, wherein said infection is caused by a medical
device, medical equipment, or an implant.

14. The method of claim 5, wherein said BALO and bacteriophage are
present in equal ratios.

18. A method of decreasing, treating or prophylaxis of a Bdellovibrio and
like organism (BALO) Gram negative prey bacterial infection in a host
cell, said method comprising administering a BALO and a bacteriophage
combination to said prey bacterial infection at a temperature of from
about 25.degree. C. to about 37.degree. C., wherein said prey bacterial
infection is Vibrio vulnificus and wherein said BALO comprises
Bacteriovorax, Peredibacter starrii, Bacteriolyticum stolpii, and
Bdellovibrio bacteriovorus, and further wherein said BALO to prey
bacterial infection in said host cell exists at a ratio of from about ten
to one, to about one to one hundred.

Description:

CROSS REFERENCE TO CO-PENDING APPLICATION

Background

[0002] Bacterial infections afflict multicellular organisms, such as, for
example, plants, animals, amphibians and humans at various stages of
development. Among children, the elderly and other immunocompromised
individuals, bacterial infections can be especially dangerous. Ear,
sinus, skin, wound, food-borne and respiratory infections are relatively
common, especially in children and are among the leading cause of visits
by children to physicians. In circumstances where an individual suffers
from chronic bacterial infections and are given conventional
antibacterial medicaments, he or she may be at greater risk of developing
bacteria that are resistant to the conventional medicaments. In fact,
bacteria that are prone to becoming resistant to antibiotics are known to
cause ear infections, nose, throat and skin infections.

[0003] Therefore, there is a need for novel medications, therapies,
methods and approaches to treating bacterial infections.

[0004] Many diseases are caused by bacterial infections. Bacteria cause up
to twenty-five percent of upper respiratory tract infections. Streptocci
bacterial are responsible for almost all cases of strep throat in the
United States. Otitis media or middle ear infections are the most common
bacterial infection in children in the United States. By age three,
two-thirds of children in the United States have had at least one episode
of ear infection and the other one third has had three or more episodes.
Common lower respiratory tract infections caused by bacteria include
pneumonia and bronchitis. Tuberculosis is another common bacterial
infection afflicting over 10 million people in the United States.

[0005] Infectious diarrhea continues to be a leading cause of morbidity
and mortality in the world. Most causes of diarrhea are viral in origin;
however, bacteria remain an important cause of diarrhea. Other bacterial
infections include salmonella, shigella, Escherichia coli, and skin
infections. Bacterial skin infections include impetigo, boils,
carbuncles, cellulitis, and burn complications.

[0006] The present invention relates to antimicrobial agents and methods
that have, for example, prophylactic and inhibitory activity with respect
to bacterial infections. The invention also relates to the use of such
antimicrobial agents and methods to inhibit, control, provide
prophylactic support or treat diseases and disorders related to bacterial
infections. More particularly, the present invention provides alternative
treatments using alone or in combination live, and or dead antibacterial
agents for use, for example, in treating bacterial infections. Even more
particularly, the present invention relates to the inhibition,
therapeutic and prophylactic preparations comprising certain isolates of
the bacterial group Bdellovibrio and like organism (BALO) in combinations
of various percentages with certain viruses that kill bacteria
(bacteriophages). In particular, the invention provides in one aspect,
prophylactic and therapeutic compositions comprising certain Bdellovibrio
and like organism (BALO) strains, acting in combination with
bacteriophage against, for example, infections of animals and humans
caused by some Gram negative pathogenic bacteria including, for example
Acinetobacter calcoaceficus, Aeromonas hydrophila, Enterobacter
aerogenes, Escherichia coli ML-35, Pseudomonas putida, Pseudomonas spp,
Proteus mirabilis, Providencia stuartii, Salmonella, Salmonella Michigan,
Salmonella Gaminola Salmonella Montbidea, Salmonella Poona, Vibrio 01,
Vibrio vulnificus CMCP6, Vibrio vulnificus M06, Vibrio sp., Vibrio
parahaemolyticus P5, and the like. The foregoing needs are met, to an
extent, by the present invention, wherein in one aspect a method is
provided of inhibiting, decreasing , treating, providing prophylactic
support, or preventing a bacterial infection in a host subject comprising
administering a therapeutically effective amount of a live Bdellovibrio
and like organism (BALO) and an active bacteriophage to a Gram-negative
bacteria-infected host subject. The Bdellovibrio and like organism (BALO)
may comprise, for example Bdellovibrio bacteriovorus, Bacteriovorax
stolpii, Bacteriovorax starrii, Bacteriovorax marinus, Bdellovibrio
bacteriovorus HD 100, Bdellovibrio bacteriovorus BD-610, Bacteriovorax
Phylotype X, Bacteriovorax Phylotype IX, Bacteriovorax Phylotype V,
Bacteriovorax Phylotope III, Bacteriovorax litoralis, and the like.

[0007] The BALO Gram negative prey bacteria may comprise Acinetobacter
calcoaceficus, Aeromonas hydrophila, Enterobacter aerogenes, Escherichia
coil ML-35, Pseudomonas putida, Pseudomonas spp, Proteus mirabilis,
Providencia stuarlii, Salmonella, Salmonella Michigan, Salmonella
Gaminola Salmonella Montbidea, Salmonella Poona, Vibrio 01, Vibrio s
CMP6, Vibrio vulnificus M06, Vibrio sp., Vibrio parahaemolyticus P5 and
the like. Prophylaxis or treatment may also be achieved by including in
the preparation a number of, for example, Bdellovibrio and like organisms
(BALO's) in various combinations with bacteriophage strains, each having
different specificities for the target prey bacteria giving the
preparation an overall total effectiveness against many more strains than
the individual bacteriophages or individual BALO. The phage panel may
include one or more bacteriophage strains which are effective against a
broad spectrum of bacteria so that the bacteriophages in the composed
preparation have overlapping effectiveness, with some specific bacteria
being targeted by multiple bacteriophages, thus helping to minimize any
development of resistance. Individual strains of the target bacterial
species may therefore be killed by one or more of bacteriophages making
up a composition or preparation.

[0009] Further examples of bacteriophage strains useful against Vibrio
vulnificus include, for example, 152A-2, 152A-8, 152A-9 152A-10, 153A-5,
153A-7, 153A-8, 154A-8, 154A-9, 108A-9, 110A-7, and 7-8a. Phage CK-2 was
isolated from estuarine mud sediment. Phage CB1 was isolated from mud
sediment, and phage EJc was isolated from oysters together with Phages
1a, 2a, 3a, 4a, 4b, AOIA-D, CKIA-B, and CKIF-G. Bacteriophages, SSP5 and
SSP6 which were classified as members of the myoviridae and Siphoviridae
families respectively have been reported to have potential to control
Salmonella.

[0011] The combination of BALO and bacteriophage may exist in various
concentrations, percentages, and ratios. The combination may also exist
in equal ratios.

[0012] The bacterial infection may comprise a Gram negative bacterium in
plants, animals, fish, amphibians, mammals, or for example humans.
Bacterial infections may comprise, for example, sinus, respiratory,
renal, skin, wounds, or ear infections of the outer or inner ear canal.
In the case of such ear infections, the administration of the prophalytic
agent or treatment may be accomplished via droplets, creams, injection,
oral suspensions, and via other administrations known in the art. Other
bacterial infections include, for example, skin infections, burn
infections, eye infections, urinary tract, and gastrointestinal
infections. Administration of the prophylactic or treatment agent may be
accomplished via injection, oral administration, creams, or suspensions,
creams or by other means known in the art. Synergistic effects have been
found for the combination of Bdellovibrio and like organisms with viruses
in the killing of pathogens that may cause these infections.

[0013] Unlike antibiotics and antiviral agents that bacteria and viruses
respectively can develop resistant to, those pathogens that are
susceptible to Bdellovibrio and like Organisms (BALOs) are not known to
develop resistance to them. Although some bacteria have been reported to
develop resistance to viruses, should this happen the Bdellovibrio and
like Organisms (BALOs) should continue to reduce the pathogens but
perhaps not as rapidly as the combination with the bacteriophage.

[0014] In embodiments, one aspect of the proposed invention shows that the
Bdellovibrio and like Organisms (BALOs) and bacteriophage are beneficial
as alternative prophylactic or therapeutic agents for reducing
Gram-negative bacterial infections in animals and humans. The prey
bacteria may comprise, for example, Acinetobactercalcoaceticus, Aeromonas
hydrophila, Enterobacter aerogenes, Escherichia coli ML-35, Pseudomonas
putida, Pseudomonas spp, Proteus mirabilis, Providencia stuarfii,
Salmonella, Salmonella Michigan, Salmonella Gaminola Salmonella
Montbidea, Salmonella Poona, Vibro 01, Vibrio s CMP6, Vibrio vulnificus
M06, Vibrio sp., Vibrio parahaemolyticus P5 and the like. Achievement of
this aspect would introduce a novel and alternative mode of treating some
of the major human Gram negative bacterial pathogens such as, for
example, Vibrio spp., E. coli, Pseudomonas, Proteus mirabilis,
Providencia stuartii, Salmonella, and improve the recovery rate for these
diseases. It has also been suggested that periodontal disease therapy
could be assisted by the application of Bdellovibrio and like Organisms
(BALOs) specifically to reduce the levels of Gram-negative pathogens in
the oral cavity.

[0015] There has thus been outlined, rather broadly, certain embodiments
of the invention in order that the detailed description thereof herein
may be better understood, and in order that the present contribution to
the art may be better appreciated. There are, of course, additional
embodiments of the invention that may be described below and which may
form the subject matter of the claims appended hereto. The present
invention therefore relates to the fields of medicine, cell biology,
mircrobiology biochemistry, and bacteriology.

DESCRIPTION

Brief Description of the Drawings

[0016] FIG. 1 shows the kinetics of the lysis of prey cells over time in
test with Bacteriovorax (Bx) and/or bacteriophages and control (with
either BALO predators or no BALO predators). Error Bars are standard
error from three independent experiments.

[0017] FIG. 2 show the growth dynamics of Bacteriovorax and bacteriophage
on prey over a forty hour period as measured by plate counts. Broken
lines designate phage count; solid lines represent Bacteriovorax counts.
Fl is the flask with both Bacteriovorax and phage. F2 and F3 are the
microcosms consisting of vibrio vulnificus with Bacteriovorax and phage
respectively.

[0018] FIG. 3 shows Transmission Electron Microscopy (TEM) micrographs
representing the predation of bacteriophages and Bacteriovorax on prey
vibrio vulnificus. The arrow in FIG. 3A points to a prey cell infected by
phage CK2. The star indicates a cell infected by Bacteriovorax to form a
bdellobast (osmotically stable structure in which the prey cell becomes
rounded) and no phage is seen inside. FIG. 3B shows Bacteriovorax and
phage both inside a vibrio vulnificus cell. Additional evidence showing
Bacteriovorax and phage are able to infect the same cell. The results
showed that Bacteriovorax and phage were able to infect vibrio vulnificus
cells separately (FIG. 3A & B) and jointly (FIG. 4).

[0019] FIG. 4 shows Bacteriovorax and phage infecting vibrio vulnificus
cells at the same time. Electron microscopy also confirmed that the
Bacteriovorax, was effective at infecting the prey, vibrio vulnificus.
Micrographs suggest that competition exists between Bacteriovorax and
phages for the source of food as they both are able to prey on the same
bacterium. Here we report a novel finding that Bacteriovorax and
bacteriophages are able to infect and reside in a single prey cell
resulting from dual infection.

Definitions

[0020] "Amount sufficient" as used herein refers to an amount that when
placed in contact with a Gram negative bacteria suppresses the growth or
reproduction of the bacteria when compared to the absence of the BALO and
bacteriophage combination.

[0021] "Antibacterial agent" as defined herein refers to an agent or
substance that destroys or suppresses the growth or reproduction of
bacteria by interfering with protein synthesis, nucleic acid synthesis,
or plasma membrane integrity; or by inhibiting critical biosynthetic
pathways in the bacteria. Such agents may be administered in
concentrations that are safe for the host and can be used as
chemotherapeutic agents to prevent or treat bacterial infections.

[0022] "Attack Phase" as used herein refers to free-swimming BALO cells
propelled by a polar flagellum at high velocities which aids the BALO in
efficiently attacking a suitable bacterium that will serve as the
substrate for its feeding and growth. The attack phase BALO cell ceases
being by the penetration of the BALO cell through the outer membrane
layer of the substrate cell and its subsequent loss of mobility.

[0023] "Safe and effective amount" as used herein refers to an amount that
is effective enough to inhibit, provide prophylactic support, reduce the
bacterial cell proliferation, or provide treatment in animals, mammals,
and more particularly, in humans without severe side effects.

[0024] The term "pharmaceutical carrier" means one or more compatible
solid or liquid filler diluents or encapsulation substances which are
suitable for administration to those suffering from disease.

[0025] "Bacteria" as defined herein refers to a domain of life existing as
small unicellular microorganisms that commonly reproduce by cell division
(fission) and are contained within a cell wall. They are a natural
component of the human body, particularly on the skin, mouth and
intestinal tract. Many are beneficial to the environment and living
organisms, but some are the cause of many infectious diseases. Infectious
bacteria enter the body through torn tissues, the openings of the nose,
mouth, lungs, contaminated food, feces, oral fecal contact and can
provoke inflammation. "Bacteria" as used herein are neither plants nor
animals. Bacteria are living things that belong to a group all by
themselves. They are small, single cell organisms called prokaryotes that
do not contain a nucleus and are widely distributed in nature, some in
very large numbers because they can quickly multiply under suitable
conditions. There are many different kinds of bacteria that are separated
into different types and groups, each group and sub group having its very
own unique qualities. Bacterial can cause myriad diseases. Bacteria that
cause disease are called pathogenic bacteria. Bacteria can cause disease
in animals, and in plants. Some bacteria attack only one host plant or
animal while other bacteria can attack or infect many types of hosts.
There is also a great diversity in where bacteria can grow. Some types of
bacteria grow best in cool, damp places like in the soil or in a pond
while others can grow in hot places like in hot water heaters or near
undersea volcanoes. There's even a species of bacteria that can withstand
blasts of radiation 1000 greater than would kill a human. No matter where
you look, whether on the ground, in your water, or in your stomach,
bacteria are there.

[0026] "Bacteriophage" (phage) as used herein are obligate intracellular
parasites that multiply inside a host bacterium by making use of some or
all of the host bio-machinery. A bacteriophage is a virus that is
parasitic in bacteria. There are many similarities between bacteriophages
and animal cell viruses.

[0027] At one time it was thought that the use of bacteriophage might be
an effective way to treat bacterial infections, but it soon became
apparent that phage were removed from the host body and thus, were
thought to be of little clinical value. However, recently, new interest
has developed in the possible use of bacteriophage for treatment of
bacterial infections and in prophylaxis. In addition, bacteriophage have
diagnostic use for the identification of pathogenic bacteria (phage
typing). Although phage typing is not used in the routine clinical
laboratory, it is used in reference laboratories for epidemiological
purposes.

[0028] Although different bacteriophages may contain different materials
they all contain nucleic acid and protein. Depending upon the phage, the
nucleic acid can be either DNA or RNA but not both and it can exist in
various forms. The nucleic acids of phages often contain unusual or
modified bases. These modified bases protect phage nucleic acid from
nucleases that break down host nucleic acids during phage infection. The
size of the nucleic acid varies depending upon the phage. The simplest
phages only have enough nucleic acid to code for three to five average
size gene products while the more complex phages may code for over one
hundred gene products.

[0029] The number of different kinds of protein and the amount of each
kind of protein in the phage particle will vary depending upon the phage.
The simplest phages have many copies of only one or two different
proteins while more complex phages may have many different kinds. The
proteins function in infection and to protect the nucleic acid from
nucleases in the environment.

[0030] Probably every known bacterium is subject to infection by one or
more viruses or "bacteriophages" as they are known. Extensive research
concerning phages has been done on the phages that attack E. coli,
especially the T-phages and phage lambda.

[0031] Like most viruses, bacteriophages typically carry only the genetic
information needed for replication of their nucleic acid and synthesis of
their protein coats. When phages infect their host cell, the order of
business is to replicate their nucleic acid and to produce the protective
protein coat. But they cannot do this alone. They require precursors,
energy generation and ribosomes supplied by their bacterial host cell.

[0032] Most bacteria, specifically pathogenic bacteria, have associated
bacteriophages that can take over the bacterial cellular machinery and
lyse the bacterial cell to release new phage particles. As such, there
was much interest in using "bacteriophage therapy" as a means to treat,
prevent and cure infection since their discovery in the early 1900's.
However the advent of antibiotics halted the majority of this research
until the past decade, which has seen renewed interest in phages due to
the increasing problem of antibiotic resistant bacteria. Although phage
therapy does show some promise, it has limitations in that bacteria are
only susceptible to infection by phage at discrete stages of growth.

[0033] Another important issue with regard to bacteriophages is that they
can kill large populations of bacteria relatively quickly. This can be
important when you are intentionally trying to produce large numbers of
bacteria. For example, when cheese is made, we grow bacteria in the milk
to give cheese its' distinctive flavor and texture. If the milk gets
infected with a bacteriophage, the culture may die out before the cheese
fermentation process is completed. When this happens, the cheese
manufacturers have vats of partially cultured milk which is not cheese,
and which usually has to be thrown away. The cheese manufacturers
sometimes refer to this as a vat of cheese that has been "phaged out";
that is, the starter culture to make the cheese has been destroyed by a
bacteriophage.

[0034] Bdellovibrio and like organism (BALO) Gram negative prey infection
host as used herein refers to Gram negative bacterium that serve as
viable and susceptible prey or parasitic victims of Bdellovibrio and like
organism (BALO), resulting in the Bdellovibrio and like organism (BALO)
feeding on the host cell's proteins and nucleic acids and the complete
lysis of the invaded host cell and the release of BALO progeny.

[0035] "Bdellovibrio growth phase" as referred to herein mean the period
when the BALO penetrate through its prey cell wall and begins to feed and
grow on its prey. This phase requires the parasitism of a suitable host
cell. It is during the Bdellovibrio growth phase when the BALO cells
grows inside its prey and multiplies producing new cells. The attacker
cells are not especially particular about the prey, except that it must
be of the Gram negative type (i.e. having a thin cell wall and
characteristic outer membrane.) When the BALO cell enters the host
bacterium it dies and bloats into a spherical shape called a bdelloplast.
Generally, the host cell loses its structural framework because it is
being eaten from the inside. The growing BALO cell is now considered a
parasite and continues to elongate into a filament. When the nutrients
(proteins, lipids, structural polymers, RNA, and DNA, etc.) are exhausted
from the host cell body, the filament partitions into the smaller attack
phase cells and are released into the environment.

[0036] "Therapeutically Effective Amount" as used herein means an amount
useful in healing, prophylaxis or curing certain patients identified in
the specification. It is not dependent on the product having an effect in
a living being, such as curing disease but, for example, to broadly claim
a pharmaceutical composition with a wide range of effects.
"Therapeutically Effective Amount" may also be used to represent a
pharmaceutical or other composition with a wide range of effects. Those
effects do not necessarily include curing diseases in plants, animals,
humans, or other species, but may also, for example, comprise amounts for
the prevention, inhibition, prophylaxis, or reduction of a condition or
disease. The amount may indicate that the claimed pharmaceutical product
has utility in the treatment of a disease where such treatment may have
an effect on the "healing" or "curing" of the disease in patients within
the class covered by the present invention.

[0037] The definition of "pharmaceutically acceptable" as used herein is
meant to encompass any carrier, which does not interfere with the
effectiveness of the biological activity of the active ingredient and
that is not toxic to the host to which it is administered.

[0038] "Gram positive and Gram negative" as used herein refers to how a
bacteria reacts to the Gram stain technique. If it takes the initial
stain, it will be purple and be considered Gram positive. If it doesn't
take the initial stain, it will be pink and Gram negative. The difference
is in the outer casing of the bacteria. A Gram positive bacterium will
have a thick layer of peptidoglycan (a sugar-protein shell) that the
stain can penetrate. A Gram negative bacterium has an outer membrane
covering a thin layer of peptidoglycan on the outside. The outer membrane
prevents the initial stain from penetrating.

[0039] "Prophylactic treatment", "prophylactic effects", "prophylactic
support" or "prophylaxis" as used herein, means measures to protect a
person from symptoms or conditions of a disease to which he or she has
been, or may be expressed.

[0040] Bdellovibrio and Like Organisms (Balo's)

[0041] The Bdellovibrio and like Organisms are extremely small bacteria
with the unique property of being predators of other Gram-negative
bacteria. In the presence of viable and susceptible bacteria a BALO cell
physically attacks a prey cell, attaches to its surface, penetrates the
cell wall, and multiplies within the periplasmic space of its prey. These
minute assassins have a peculiar lifestyle: they swim around at high
speed and after collision with a susceptible prey bacterium; they attach
to their prey and enter its periplasm. BALO and its progeny degrade and
consume the cellular constituents. The life cycle of BALO alternates from
the mobile, non-growing attack phase and the growth phase. BALO's are
found in a wide variety of ecosystems but particularly in sewage and
other areas densely populated with bacteria. BALO were not found in frog
or crab intestinal tracts, but have been recovered from the gut of
humans, horses, and chickens. The occurrence and distribution are
influenced by factors such as, for example, temperature, salinity,
habitat, prey polulation density, and pollution. Salinity levels have
been found, for example, from about 0.0 to over 1.0 percent. Predatory
activity of Bdellovibrio has sometimes found to be inhibited by the
presence of detergents, heavy metals, and pesticides.

[0042] Some Bdellovibrio and like Organisms (BALOs) are halophilic.
Halophilic or salt-loving bacteria are indigenous organisms of salt
packs, brines, or bodies of salt water. They thrive in concentrations of
salt, for example, of from about one percent, to about fifteen percent
salt or from about 0.5 to about 2.5 M salt concentration. Salt
requirements and salt tolerances of many species vary according to growth
conditions such as media composition and temperature. Halophilic bacteria
have been found all over the world from the Dead Sea to the Great Salt
Lake, and in all of the earth's oceans. They are also found in the saline
soils of Antarctica, the tropics and desert environments. Most species
maintain their intracellular ionic concentrations at low levels while
synthesizing or accumulating organic solutes to provide osmotic
equilibrium of the cytoplasm with the surrounding medium.

[0043] Bacteriovorax are members of the saltwater genus Bacteriovorax,
formerly known as the marine Bdefiovibrio, are obligate predatory
bacterium that prey selectively on other Gram-negative bacteria.

[0044] Bacteriolyticum stolpii Bacteriolyticum stolpfi is an obligate
predatory bacterium that preys upon a wide variety of susceptible
Gram-negative bacteria. Bacteriolyticum stolpfi has an optimal growth
temperature of from about 29 to about 31° C.

[0045] Peredibacter starrii is another strain of the family of
Bacteriovoracaceae predatory bacteria. Peredibacter starrii is found in
freshwater and in the soil. Peredibacter starrii is vibriod shaped
bacteria about 0.4 to about 0.5 μm in length.

[0046] Bdellovibrio and Like Organisms (Balos) Method of Attack

[0047] Bdellovibrio (Bd), which literally means "curved leech", and like
organisms makes their living by attacking and devouring other bacteria,
and are found in diverse environments such as marine and fresh waters,
sewage, and soil. Bacteria of this type are characterized by two distinct
stages in their life cycle, a predatory "attack" phase, and a parasitic
"growth" phase.

[0048] During the attack phase, the cell has a curved rod shape of
approximately 1.4 micrometers in length, and has a single whip-like
projection called a flagellum. This bacterial flagellum rotates like a
corkscrew to propel the bacterium at a rate of 100 microns per second.
Considering the size of the cell, this corresponds to an incredible 70
body lengths per second! These highly motile attack phase cells have no
sense of direction; instead, the flagellum propels the cell in whatever
direction it happens to be pointing. Finding prey, therefore, is limited
to "bumping" into a suitable prey cell that just happens to be in the
right place at the wrong time. When a prey cell is encountered, the BALO
continues to rotate and bore its way through the outer cell wall and into
the prey where it lodges in the periplasmic space. Once inside, the
attacker loses its flagellum, feeds upon the prey cytoplasm, grows and
elongates and prepares for the multiplication process.

[0049] Prey Species of Bdellovibrio and Like Organisms (Balos)

[0050] Acinetobacter calcoaceticus is a pleomorphic aerobic Gram-negative
bacillus commonly found in hospital and on hospital patients in the
United States and cultured from the patients' sputum or respiratory
secretions, wounds, and urine. Acintobacter has been found to colonize
irrigating and intravenous solutions. Acinetobacter infections usually
involve the respiratory tract, peritoneal fluid, and the urinary tract.
Prolonged hospitalization or antibiotic therapy predisposes to
Acinetobacter colonization. Acinetobacter have been found to be
multi-drug resistant Few antibiotics are active against this organism.
Acinetobacter is generally sensitive to Meropenem, Colistin, Polymyxin B,
Amikacin, Rifampin, Minocycline, and Tigecycline. Cephalosporins,
macrolides, and penicillins have little or no effect on Acinetobacter
activity and may predispose to Acinetobacter colonization.

[0051] Aeromonas hydrophila (A. hydrophila) is found present in freshwater
environments and also in brackish water. The bacterium is capable of
causing illness in fish, amphibians and humans. Humans typically acquire
infections through an open wound or ingestion of the organism in food or
water. Aeromonas hydrophila may cause gastroenteritis in healthy persons
or septicemia in persons with compromised immune systems or various
malignancies. Aeromonas hydrophila size ranges from about 1.0 to about
3.5 micrometers in length and has a diameter of from about 0.3 to 1.0
micrometers. Aeromonas hydrophila can grow at temperatures of from about
4° C. to about 37° C. with an optimal growth at about
28° C. In humans, Aeromonas hydrophila is typically transmitted
through oral fecal contact, contact with contaminated water, food, soil,
feces, and ingestion of contaminated fish or reptiles. Most common is
infection through an open wound in contaminated water. The microbe is
resistant to penicillin, ampicillin, carbenicillin, and ticarcillin but
susceptible to cephalosporins, aminoglycosides, carbapenems,
chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, and the
quinolones.

[0052] Enterobacter aerogenes is a Gram-negative bacillus belonging to the
Enterobacteriaceae family. Enterobacter aerogenes is another
infection-causing bacteria that is widely distributed in nature,
occurring in fresh water, soil, sewage, plants, vegetables, and human
feces. Enterobacter aerogenes can cause, for example, burn, wound, skin,
endocarditis, soft tissue, ophthalmic, urinary tract infections. It may
also be responsible for septicemia and menigitis. Enterobacter aerogenes
ranges from about 0.6 to about 1.0 micrometers wide and from about 1.0 to
about 3.0 micrometers in length. Optimal growth occurs at from about
35° C. to about 37° C.

[0053] Escherichia coli is another prey bacteria for Bdellovibrio and like
Organisms (BALOs) which live in the intestines of people and animals.
Most varieties of E. coli are harmless or cause brief diarrhea. Some more
serious strains can cause severe, bloody diarrhea and abdominal cramps,
followed by organ system damage, such as kidney failure. Exposure may
come from contaminated water or food such as raw vegetables and under
cooked ground beef. Young children and older adults can develop
life-threatening kidney failure such as hemolytic uremic syndrome.

[0054] Pseudomonas putida are Gram-negative bacteria prey for BALO's.
Pseudomonas putida are fluorescent, aeorbic, non spore forming, oxidase
positive bacteria. Having one or more polar flagella, they are motile
organisms. They can be found in moist environments, such as soil and
water, and grow optimally at room temperature. Certain strains have the
ability to grow on and break down many dangerous pollutants and aromatic
hydrocarbons such as toluene, benzene, and ethylbenzene. Pseudomonas
putida can also be used in petroleum plants to purify fuel. Pseudomonas
putida play a huge role in bioremediation, or the removal or
naturalization of soil or water contaminants. They can degrade toluene,
xylene, and benzene, which are all toxic components of gasoline that leak
into the soil by accidental spills. Other strains can convert styrene,
better known as packing peanuts, which do not degrade naturally, into a
biodegradable plastic. Due to the fact that Pseudomonas putida can use
styrene as its only source of carbon and energy, it can completely remove
this toxic chemical over time from the environment. Pseudomonas putida
can also turn Atrizine, a herbicide that is toxic to wildlife, into
carbon dioxide and water. Being a non-pathogenic bacterium, there has
been only a few instances where Pseudomonas putida has infected humans.
For the most part, it has been associated with immunocompromised
patients, causing septicaemia, pneumonia, urinary tract infections,
nosocomial bacteremia, septic arthritis, or peritonitis. They are able to
protect plants from pests, promote plant growth, and clean up organic
pollutants found in soil and water.

[0055] Pseudomonas spp is a Gram-negative, oxidase-positive, motile rod
bacterium which grows yellow-green iridescent colonies. Pseudomonas spp
infections can develop in many places on the human body including skin,
subcutaneous tissue, bone, ears, eyes, urinary tract, and heart valves.
The most serious infections occur in debilitated persons with compromised
immune systems resulting from other disease or therapy. Pseudomonas spp
occurs most often in hospitals, where it can be found in sinks,
antiseptic solutions, and urine receptacles and by cross infection via
the hands of hospital personnel. When infection is localized and
external, treatment with 1% acetic acid irrigations or topical agents
such as polymyxin B or colistin is sometimes effective. Other antibiotics
used include amikacin, tobramycin and gentamicin. Several penicillins,
including carbenicillin, ticarcillin, piperacillin, mezlocillin, and
azlocillin, are active against Pseudomonas.

[0056] Proteus mirabilis is part of the normal flora of the human
gastrointestinal tract. It can also be found free living in water and
soil. When this organism, however, enters the urinary tract, wounds, or
the lungs it can become pathogenic. Proteus mirabilis commonly causes
urinary tract infections and the formation of stones. Proteus mirabilis
is characterized by its swarming motility, its ability to ferment
maltose, and its inability to ferment lactose. Proteus mirabilis has the
ability to elongate itself and secrete a polysaccharide when in contact
with solid surfaces, making it extremely motile on items such as medical
equipment. The most common infection involving Proteus mirabilis occurs
when the bacterium moves to the urethra and urinary bladder. Although
Proteus mirabilis is mostly known to cause urinary tract infections, the
majority of urinary tract infections are due to E. coll. Urinary tract
infections caused by P. mirabilis occur usually in patients under
long-term catherization. The bacteria have been found to move and create
encrustations on the urinary catheters. The encrustations cause the
catheter to block. Symptoms for urethritis are mild including frequency
of urination and pyuria (presence of white blob cells in the urine).
Cystitis (bladder infection) symptoms are easier to distinguish and
include back pain, concentrated appearance, urgency, hematuria (presence
of red blood cells in the urine), and suprapubic pain as well as
increased frequency of urination and pyuria.

[0057] Pyelonephritis (kidney infection) can occur when the bacteria
migrates from the lower urinary tract. Although it is seen as a
furtherance of infections, not all patients have the symptoms associated
with urethritis and cystitis. Pyelonephritis is marked by nausea and
vomiting.

[0058] Proteus mirabilis also can enter the bloodstream through wounds.
This happens with contact between the wound and an infected surface. The
bacteria induce inflammatory response that can cause sepsis and Systemic
Inflammatory Response Syndrome (SIRS). SIRS has a mortality rate between
twenty and fifty percent.

[0059] Proteus mirabilis can also, though less common, colonize the lungs
and causes pneumonia. This is the result of infected hospital breathing
equipment. Symptoms for pneumonia include fever, chills, chest pain,
rales, and cough. Prostatitis can occur as a result of Prostatitis
mirabilis infection, causing fever, chills, and tender prostate in men.
Proteus mirabilis infections can be treated with broad-spectrum
penicillins or cephalosporins except in severe cases. It is not
susceptible to nitrofurantoin or tetracycline and has experienced
increased drug resistance to ampicillin, trimethoprim, and ciprofloxin.
In cases with severe stone formation, surgery is necessary to remove the
blockage. Proteus mirabilis is part of the normal flora of the
gastrointestinal tract, and as a result the bacterium enters the urinary
tract or infects medical equipment by the fecal route. Consequently,
prevention includes good sanitation and hygiene, including proper
sterilization of medical equipment. It is also suggested that patients
not requiring catherization should not receive catherization, despite its
convenience for the caretaker.

[0060] Providencia sturtii is a Gram negative, flagellated motile
bacterium. It is an aerobic micro organism that is best grown at about
37° C. The bacterium is found in sewage and contaminated waters.
It is also found in humans and animals. They are known to cause urinary
tract infections, traveler's diarrhea and have been isolated from wounds
caused by third degree burns. It can also reside in the gastrointestinal
tract of humans and animals.

[0061] Salmonella Agona. Salmonella is a Gram negative, rod-shaped bacilli
that can cause diarrheal illness in humans. Most persons infected with
Salmonella develop diarrhea, fever, and abdominal cramps twelve to
seventy-two hours after infection. Infection is usually diagnosed by
culture of a stool sample. The illness usually lasts four to seven days.
Although most people recover without treatment, severe infections may
occur. Infants, elderly persons, and those with impaired immune systems
are more likely than others to develop severe illness. When severe
infection occurs, Salmonella may spread from the intestines to the
bloodstream and then to other body sites and can cause death unless the
person is treated promptly with antibiotics. Every year, approximately
35,000 cases of food poisoning caused by the salmonella bacterium are
reported. Because most mild cases are never reported, the actual number
is probably quite larger. Children are the most likely to get
salmonellosis. Young children, older adults, and people with impaired
immune systems are the most likely to have severe infections. They are
microscopic living creatures that pass from the feces of people or
animals to other people or other animals. The rod-shaped enterobacterium
has a diameter of from about 0.5 to about 1.5 micrometers and a length of
from 2.0 to about 5 micrometers.

[0062] Salmonella can survive for weeks outside a living body. They have
been found in dried excrement after more than two and a half years.
Salmonella are not destroyed by freezing. Ultraviolet radiation and heat
accelerate their demise; they perish after being heated to 55° C.
(131° F.) for one hour, or to 60° C. (14 ° F.) for
half an hour. With poultry, cattle, and sheep frequently being agents of
contamination, salmonella can be found in food, particularly meats and
eggs. To protect against Salmonella infection, it is recommended that
food be cooked for at least ten minutes at 75° C. (167° F.)
so that the center of the food reaches this temperature. Salmonella
bacteria can survive several weeks in a dry environment and several
months in water; thus, they are frequently found in polluted water,
contamination from the excrement of carrier animals being particularly
important. Aquatic vertebrates, notably birds and reptiles, are important
vectors of Salmonella.

[0063] Salmonella Poona has been associated with outbreaks in cantaloupes
and turtles. Symptoms included nausea, vomiting, diarrhea, abdominal
cramps, and fever; the duration of symptoms was three to twelve days.

[0064] Vibrio Cholerae 01 is one of two strains associated with epidemic
cholerae. Vibrio 01, the bacterium that causes the disease cholera,
controls virulence factor production and biofilm development in response
to two extracellular quorum-sensing molecules, called autoinducers. The
organism is a comma-shaped, Gram-negative aerobic bacillus whose size
varies from one to three micrometers in length by 0.5-0.8 micrometers in
diameter.

[0065] Vibrio s CMP6 is one of the few vibrio vulnificus strains of which
the full genome has been sequenced.

[0066] Xylella fastidiosa, the pathogen causes Pierce's Disease, which has
been a national issue for the viticulture industry. Xylella fastidiosa
clogs a plant's xylem and effectively shuts down its ability to take in
water and nutrients. The disease is spread naturally through the feeding
activities of leafhopper vectors (Mizell, 1990).

[0069] Vibrio parahaemolyticus P5 is one of the most widely used prey
bacteria for isolation of halophilic BALOs.

[0070] In embodiments of the present invention, a preparation is provided
comprising a therapeutically effective amount of a Bdellovibrio and like
organism (BALO) and a bacteriophage combination introduced into a prey
bacteria-infected subject.

[0072] In an embodiment of the present invention, a composition is
provided comprising a therapeutically effective amount of one or more
strains of a Bdellovibrio and like organism (BALO) and one or more
bacteriophage introduced in to a Bdellovibrio and like organism (BALO)
prey bacteria-infected host subject. The BALO and the bacteriophage
combination may exist, for example, in equal proportion or in various
ratios or percentages.

[0075] A bacteriophage is a virus that is parasitic in bacteria. A
bacteriophage uses the bacterium's energy and processes to produce more
phages until the bacterium is destroyed and the new phage particles are
released to invade surrounding bacteria. At one time it was believed that
the use of bacteriophage might be an effective way to treat bacterial
infections, but it was thought that phage are quickly removed from the
body and thus, some research in the area was abandoned. Recently, new
interest has developed in the possible use of bacteriophage for treatment
of bacterial infections and in prophylaxis. There is growing evidence
that bacteriophage will be used in clinical medicine in the future.

[0076] Although different bacteriophages may contain different materials
they all contain nucleic acid and protein. Depending upon the phage, the
nucleic acid can be either DNA or RNA but not both and it can exist in
various forms. The nucleic acids of phages often contain unusual or
modified bases. These modified bases protect phage nucleic acid from
nucleases that break down host nucleic acids during phage infection. The
size of the nucleic acid varies depending upon the phage. The simplest
phages only have enough nucleic acid to code for three to five average
size gene products while the more complex phages may code for over one
hundred gene products.

[0078] In embodiments, the methods, therapies, preparations and
compositions of the instant invention have been performed to evaluate
their individual and combined effect of combinations of Bdellovibrio and
like organism (BALO) with varying percentages of bacteriophage in
treating bacteria susceptible to strains of BALO and phage. One strain
was tested in rabbits by the experimental ilial loop technique against
Escherichia coli. The Bdellovibrio and like organism (BALO) strain Vibrio
vulnificus in oysters was also tested for effacacy. Preliminary results
suggest that when using phages or BALO, limited success was achieved.
Based on modeling and laboratory results, we believe it more effective to
use Bdellovibrio and like organism (BALO) in combination with phages to
treat pathogens in infections. Synergistic effects have been realized and
are shown in the examples of the instant application. Theoretically and
experimentally, Bdellovibrio and like organism (BALO) are able to control
a wide range of Gram negative bacteria whereas a cocktail of the
bacteriophage strains were usually used to treat infection since their
host range was narrower. We have found it to be very promising and
efficacious to treat infections with bacteriophages and Bdellovibrio and
like organism (BALO) in combination to achieve better results.

[0079] Bdellovibrio and like organism (BALO) and bacteriophages tested
have not been shown to possess the required molecular machinery to infect
humans indicating the safety of using the agents in combination to treat
human bacterial diseases. If using these biological agents to
decontaminant harmful bacteria before reaching the human body such as in
foods for human consumption, presumably, the higher dose may achieve
quicker effects. The highest dose of BALOs and phages we have tested were
about 4×108 PFU/mL, which were able to decrease by 5 logs the
Vibrio vulnificus population within 40 hours in seawater. Those of skill
in the art will determine the proper amounts, percentages,
concentrations, and dosages using standards methods. However, a range of
from about 4×106 to about 4×1010 PFU/ml for both is
reasonable.

EXAMPLES

[0080] The following Examples are provided to illustrate certain aspects
of the present invention and to aid those of skill in the art in
practicing the invention. These Examples are in no way to be considered
to limit the scope of the invention in any manner.

Prey Suspensions to Eestablish the Microcosms or For Plating For
Bacteriovorax Recovery

[0082] Prey suspensions were prepared by adding 5 mililiters of 70%
artificial sea water (ASW) (Instant Ocean, Aquarium Systems, Inc.,
Mentor, Ohio) (pH 8, salinity 22 ppt.) to Luria-Bertani (LB) culture
plates (Difco) containing an 18 hour culture of vibriovulnificus. The
colonies on the plates were suspended in the liquid and the resulting
prey suspension was transferred into a sterile tube for subsequent use.
The viable prey bacterial counts were measured by spread plating in
duplicate 0.1 mililiters of serial 10-fold diluted samples onto LB agar
plates. The plates were incubated at 37° C. for two days and
colony forming units (CFU) were counted and recorded. This or similar
procedures known to those in the art was used to prepare other BALO prey
suspensions.

Example 3

Example of Isolating Highly Efficient Predator Bacteriovorax Strains

[0083] Water samples were collected from sites in three different bodies
of water located in Florida (USA): Dry Bar in Apalachicola Bay on three
occasions, the eastern coast of the Gulf of Mexico, and Atlantic Ocean
coastal waters off northern Florida. Immediately after being transported
to the laboratory, water samples were mixed and filtered through a 0.8
micrometers (μm) filter to remove debris and larger organisms, for
example, protists. Five hundred milliliters of the filtrate was dispensed
into each of four 2 liter (L) Erlenmeyer flasks for the microcosm
enrichment experiments. To complete the microcosms for the enrichment of
Bacteriovorax, suspensions of target bacteria, Vibrio vulnificus and
Vibrio parahaemolyticus were spiked as prey into the respective flasks
described above to yield an optical density (OD) measurement of 0.7 at
600 nanometers (nm). This corresponds to approximately 5×108
cells ml-1 as enumerated by plate count on LB-rif agar plates [LB
agar with 50 μg ml-1 rifampicin]. The two control microcosms
established to monitor the OD of the prey without interference from
Bacteriovorax or other microorganisms consisted of equal volumes of prey
in sterilized environmental water. The microcosm flasks were shaken at
room temperature and monitored at 24 hour intervals through 120 hour by
OD measurements (at 600 nm) in 48-well microtiter plates by an Absorbance
Microplate Reader.

[0084] Isolation of Predominant Bacteriovorax Strains in Microcosms

[0085] Samples from the test microcosms were cultured for Bacteriovorax
using the double agar overlay technique (Williams and Falkler, 1984).
Before (pre-spike) and immediately (0 hours) after addition of the prey
bacteria, 5 milliliters of sample were inoculated into Pp20 top agar
tubes with 1 milliliter of prey bacteria (Vv or Vp) and plated onto large
culture plates (150×15 mm). At subsequent time points, samples from
the test microcosms were diluted by a series of 10-fold dilutions. One
milliliter of the dilution was inoculated into 3.5 milliliters of molten
Pp20 top agar tubes with 500 μl of prey. The contents of the tubes
were mixed and overlaid onto Pp20 bottom agar plates. The plates were
incubated at room temperature for up to 8 days. Plaque-forming units
(PFUs) were counted and recorded. Plates with approximately 30 or less
plaques were selected for further processing as these represented the
predominant culturable Bacteriovorax OTU within the microcosm at the time
of plating. Material from 80 to 100 percent of the plaques on these
plates were collected with sterile micropipette tips and each inoculated,
respectively, into a 1.5 mililiters eppendorf tube containing 50
microliters (μl) of autoclaved MiliQ water and stored at -20°
C. for subsequent Polymerase Chain Reaction(PCR) analysis.

[0086] To determine the phylotype of highly efficient predator
Bacteriovorax isolated, the tubes containing the selected Bacteriovorax
plaques were boiled at 100° C. for 20 minutes. Ten micro-liters
(μl) of the suspension was PCR amplified using Bacteriovorax specific
primers, Bac-676F (5'-ATT TCG CAT GTA GGG GTA-3') and Bac-1442R (5'-GCC
ACG GCT TCA GGT AAG-3') (Davidov, 2006) by puReTaq Ready-To-Go PCR Beads.
All amplifications were performed under the following thermal conditions:
initial denaturation at 95° C. for 3 minutes, followed by 34
cycles of 96° C. for 3 minutes, annealing at 55° C. for 1
minute, extension at 72° C. for 1 minute and a final extension at
72° C. for 7 minutes in an iCycler thermocycler. PCR products were
analyzed by electrophoresis for amplicons of approximately 760 bp,
purified with the QIAquick PCR-Purification Kit (QIAGEN) and sequenced
with Bac-676F primer. DNA sequences and homology searches were analysed
with the Basic Local Alignment Search Tool (BLAST) server from the
National Center of Biotechnology Information (www.ncbi.nlm.nih.gov).
Sequences were also analysed using the Chimera_Check, version 2.7 from
the RDP-II Web site (Cole et al., 2003).

[0087] Bacteriovorax plate counts were obtained using the double agar
overlay technique (Williams and Falkler 1984). Briefly, aliquots of
samples were 10-fold serially diluted and inoculated into 3.5 mililiters
molten Pp20 top agar (Pp20 bottom agar: 1 g L-1 Pepton, 15 g L-1 agar;
Pp20 top agar: 1 g L-1 Peptone, 7 g L-1 agar) with 500 μl Vibrio
vulnificus as prey. The contents of the tubes were mixed and overlaid
onto Pp 20 bottom agar plates. The plates were incubated at room
temperature (RT). The presence of Bacteriovorax plaques on the plates was
monitored daily for a week and plaque forming units (PFUs) were counted
and recorded within three days of their initial appearance.

Example 4

Example of Isolating Bacteriophage Strains Against Target Bacteria

[0088] The phage enrichment and isolation procedures were conducted on
both estuarine mud sediment and oysters. When using oysters for the phage
enrichment and isolation procedures, oysters were first scrubbed and
washed under running deionized water. They were then shucked, and the
oyster meat was removed for further use. Seawater at a salinity of 20
parts per trillion (ppt) was added to the oyster tissue at 1 mL/g of
oyster tissue, and the mixture was homogenized. For all experiments using
seawater, seawater was always used at 20 ppt. Fifty milliliters of either
the homogenate or sediment mud contents was mixed with 50 mililiters of
LB. The mixture was then inoculated with 1 mililiters of static overnight
starter culture(s) consisting of vibrio vulnificus strain(s) of interest.
The inoculated mixture was shaken overnight at 37° C.

[0089] After shaking overnight, the mixture was centrifuged at about
13,776×g for about 10 minutes at about 4° C. to remove
bacterial debris, oyster homogenate, and mud sediment. The supernatant
was then filtered through a 0.2 μm filter. The amplified phages in the
filtrate were further amplified by mixing 1 mililiters of the filtrate
with 1 mililiters of static overnight starter culture of vibrio
vulnificus strain(s) of interest. This mixture of phage and bacteria was
supplemented with 8 mililiters of LB and shaken overnight at about
37° C. If more than one vibrio vulnificus strain was used for the
phage enrichment procedure, the filtrate was amplified separately in each
of the vibrio vulnificus strains. After the culture was shaken overnight,
it was centrifuged at 13,776×g for about 10 minutes at about
4° C., and the resulting supernatant was filtered through a 0.2
μm filter. Approximately 20 μl of the filtrate was streaked on LB
plates, and about 4 mililiters of LB-SW soft agar inoculated with
1×107 CFU/ml of log phase vibrio vulnificus was poured onto LB-SW
plates from the least concentrated area towards the most concentrated
area of the streaked filtrate. The LB plate(s) was incubated overnight at
about 37° C.

[0090] The next day, the clearest and most isolated plaques were picked
using a Pasteur pipet. The agar plug was placed in 100 μl of BSG
containing 10 μl of chloroform and was stored overnight at about
4° C. The mixture was then centrifuged at 13,776×g for 10
minutes at about 4° C. to remove bacterial debris and agar. The
supernatant was used for a plaque purification step. The supernatant was
streaked on a LB plate, and soft agar containing 1×107 CFU/mL
of log phase vibrio vulnificus was poured over the LB plate, as described
above. The plate was incubated overnight at about 37° C., and the
clearest and most isolated plaque was again picked using a Pasteur pipet.
The agar plug was placed in 100 μl of BSG containing 10 μl of
chloroform and stored overnight at a temperature of about 4° C.
The next day, the mixture was centrifuged at 13,776×g for 10
minutes at about 4° C. to remove bacterial debris and agar. The
resulting supernatant was stored at about 4° C. for the
amplification procedure.

Example 5

Bacteriophage Amplification

[0091] Both broth and plate methods were employed for phage amplification.
The majority of phages amplified efficiently using the broth
amplification method, although certain phages amplified more efficiently
using the plate method. Thus, the broth amplification technique, which
necessitates less time for phage amplification, was utilized in certain
embodiments unless, as detailed above, phages required the plate
amplification technique for more efficient amplification.

[0092] Broth Phage Amplification Method

[0093] For the broth phage amplification method, 1 L of LB-N or LB-SW was
inoculated with 5 mililiters of static overnight starter culture of
vibrio vulnificus. The culture was shaken at about 37° C. until
the culture reached an optical density corresponding to about
2×107 CFU/mL. The culture was then infected with bacteriophage
an MOI of 0.02 and shaken at about 37° C. until a change in the
culture was observed from turbid to clear, corresponding to the phage
induced lysis of the bacteria. One milliliter of chloroform was then
added to the culture, which was shaken at about 37° C. for an
additional 15 minutes to lyse any remaining bacteria. The culture was
centrifuged at 13,776×g for about 10 minutes at 4° C. to
remove bacterial debris, and the supernatant was stored at about
4° C. for the purification procedure.

[0094] Plate Phage Amplification Method

[0095] For the plate phage amplification method, 10 mililiters of LB-N or
LB-SW broth was inoculated with a static overnight starter culture of
vibrio vulnificus at a dilution of 1:20. The culture was shaken at about
37° C. until the culture reached a density of about
2×108 CFU/mL, determined by OD600, and then about 4×107
CFU was combined with a volume of phage equivalent to an MOI of 0.5, and
the tube was vortexed. After about a 10 minutes incubation period at room
temperature, 4 mililiters of LB-SW soft agar was combined with the
phage-bacteria mixture, vortexed, and poured onto a LB-SW plate. The soft
agar overlay LB-SW plate was incubated overnight at about 37° C.
The soft agar was removed using a sterile spatula, suspended in either 5
mililiters of BSG or seawater containing 20 μL of chloroform, and
stored at about 4° C. After at least about 4 hours, the mixture
was centrifuged at 13,776×g for about 10 minutes at about 4°
C. to remove the soft agar and bacterial debris. The supernatant was
stored at 4° C. for the purification procedure.

[0096] Purification of Phage

[0097] For purification of phage, 0.2 mililiters of 20 percent (wt/vol)
polyethylene glycol (PEG) 8000, 2.5 M NaCl was added per mililiters of
phage solution, vortexed, and stored overnight at about 4° C. The
phage mixture was centrifuged at 13,776×g for about 10 minutes at
about 4° C., and the resulting supernatant was discarded. To
remove the remaining PEG in the phage suspension the mixture was
centrifuged once more for approximately 1 minute at 13,776×g at
4° C., and the remaining supernatant was removed using a Pasteur
pipet. The pellet was suspended in seawater and filtered through a 0.2
μm filter. The filtrate was stored at 4° C.

[0098] Quantification of Phage

[0099] For new phage solutions in which phage titers were unknown, the
drop titer method was initially utilized to establish an approximate
titer for each phage. The full plate titer method was utilized to
establish a more accurate phage titer for each phage in the collection.
Sterile seawater at 20 ppt was used for dilution of phage for all
quantification assays. A culture of vibrio vulnificus was grown to about
2×108 CFU/mL, and about 4×107 CFU was infected with
100 μl of serially diluted phage and vortexed. After 10 minutes
incubation at room temperature, about 4 mililiters of LB-N or LB-SW soft
agar was added and vortexed. The resulting mixture was then poured over a
LB-SW plate, and the plate was incubated overnight at about 37° C.
The next day, the plaques were counted, and titer was calculated.

[0100] General Procedures

Example 6

[0101] The Bacteriovorax present in the cultures were enumerated by
quantitative real-time PCR (qPCR) (Zheng et al., 2008). Briefly, 1
mililiters samples were removed at 4 hour intervals and genomic DNA was
extracted using the QlAamp DNA Mini kit (QIAGEN) with a final product of
100 μl eluted. Bacteriovorax specific primer set 519F
(5'-CAGCAGCCGCGGTAATAC-3') and 677R (5'-CGGATTTTACCCCTACATGC-3') was used
for quantification of the Bacteriovorax. qPCR analysis was performed by
using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad,
Hercules, Calif., USA). The qPCR reaction mixtures (25 μl) were
composed of 12.5 μl of iQ SYBR Green Supermix (Bio-Rad), 1 μl of
each primer (5 pmol μl-1) primer, 1 μl of sample DNA and 9.5
μl of MiliQ water. Thermal cycling conditions were: 2 minutes at
94° C., followed by 45 repeats of 30 sec at 94° C., 10 sec
at 62° C. and 10 sec at 72° C. Each sample was measured in
triplicate and negative controls (no template) were included. A 10-fold
dilution series of plasmid containing a fragment of the Bacteriovorax 16S
rRNA gene was used in the qPCR assay to construct the standard curve
(correlation coefficient >0.99).

[0102] The combined effect of phages and BALOs in treating of Bdellovibrio
prey bacterial infections were tested in seawater and two times in Luria
Bertani broth. The temperatures we used were, for example, from about
25° C. to about 37° C. Test parameters including optical
density readings, growth rate of the two predators and viable counts of
the prey. Morphological features of BALO and phage infections were
characterized by electron microscopy. Predator: prey ratios tested ranged
from 10:1 to 1:100. BALOs and phages were inoculated in equal number
initially and then the test cultures were monitored over about a 40 hour
period with samples examined at selective time points.

[0103] To establish experimental cultures, equal numbers of Bacteriovorax
and bacteriophages in respective suspensions were inoculated into a test
microcosm containing Vibrio vulnificus suspended in 200 mililiters
sterilized natural sea water at a predator: prey ratio of 10:1. Three
control microcosms were established to monitor the growth of Vibrio
vulnificus with Bacteriovorax and bacteriophages respectively or with no
predators. Cultures were incubated at 27° C. on a shaker for 40
hours to monitor the population dynamics between the predator and prey
and their respective abundances at selected time points. Test and control
microcosms were monitored by measurements of OD values every 4 hours.
Aliquots of samples were removed at 0, 12, 20 and 40 hours to obtain
viable counts of Bacteriovorax, bacteriophages, and prey by plating
methods described above.

[0104] To investigate and observe the combined effect of Bacteriovorax and
bacteriophages by transmission electron microscopy, approximately
1×10 8 PFU ml-1 of Bacteriovorax cluster IX and
bacteriophage CK2 were inoculated at predator-prey ratios of 1:1 into
Luria-Bertani (LB) broth containing vibrio vulnificus at late logarithmic
phase. The fresh prey culture was prepared by diluting the static
overnight starter culture 1:20 into LB broth and shaking at 37° C.
About 1.5 hour later, the shaking culture normally reached a cell density
of approximately 2×108 CFU ml-1, corresponding to late
logarithmic phase of growth. The prey bacteria were harvested by
centrifugation at 13,776×g at room temperature for about 10
minutes. The resulting pellet was suspended in seawater prior to the
addition of the predator. The mixture containing the two predators and
their prey was shaken at about 37° C. Fifty mililiters of sample
were removed after about 30 minutes, 1 hour, and 4 hours and fixed for
electron microscopy examination according to Koval and Bayer, (1997) with
modification. Briefly, samples were centrifuged for 30 minutes at
27,485×g, resuspended in 1 mililiters of 0.1 M sodium phosphate
buffer (pH 7) and centrifuged for about 20 minutes at 20,142×g. The
pellet was resuspended in 2 mililiters of 0.1 M cacodylate buffer
containing 2 percent glutaraldehyde and 1 percent formaldehyde, both
diluted from 25 percent (v/v) and 16 percent (v/v) stock solutions,
respectively. After 1 hour at about 4° C. and centrifugation at
20,142×g for about 20 minutes, the pellet was overlaid with
cacodylate buffer and an aliquot of sample was stained with uranyl
acetate and examined with a Hitachi H-7600 transmission electron
microscope.

[0105] The basic experiment described above will be adapted to evaluate
the effects of experimental and environmental variables on the predation
rate of BALOs and bacteriophages on test prey. The variables to be tested
include: i) Initial predator-prey ratios of from about one to 100 to
about ten to one, ii). Different prey bacteria (spike in single bacterium
spp. as well as consortia of multiple species, iii). Various nutrient
conditions, iv). Temperature range, v). Salinity range, and vi). pH
range.

[0106] Effectiveness of Bacteriovorax and Phage in Reducing Vibrio in
Oyster Model

[0107] Bacteriovorax and phage will be tested in an oyster model
originally designed for evaluating bacteriophages (Martin, 2005) to
investigate their effect on controlling Vibrio vulnificus in vivo.
Briefly, live, fresh oysters from Dry Bar in Apalachicola Bay where the
tested Bacteriovorax strain was originally isolated will be cleaned and
placed in a pan with autoclaved seawater aerated with a Maxi-Jet 400
aquarium pump. After overnight acclimation and treatment with Rifampicin
(Rif) (added to the seawater at a final concentration 50 μg/mL) to
kill the natural bacterial flora and potentiate infection by the Vibrio
vulnificus strain, the oysters will be experimentally infected with log
phase Rif resistant Vibrio v. FLA042 or FLA077 at a final concentration
of 1×106 CFU/mL. Equal numbers of Bacteriovorax and phage
ranging from 1×107 to 1×109 PFU/ml will be added
individually and in combination to the oysters in the seawater for
various periods of time. The oyster tissue will be harvested and
homogenized and cultured to enumerate CFU of Vibrio vulnificus Vibrio
vulnificus and PFU of Bacteriovorax and phage. The response of the two
predators will be compared by analysis of variance (ANOVA) between
groups. Real time PCR will be used as a nonculture method to enumerate
Bacteriovorax and Vibrio vulnificus to validate plate count results.

[0108] Each of the strains will be grown and harvested as described in the
above section. A purified suspension of each BALOs or bacteriophage
isolate will be added to the water in the experimental holding tank for
the rif treated oysters to achieve a final BALO/bacteriophage
concentration of 109 plaque-forming units ml-1.

[0109] To examine potential detrimental effects of Bacteriovorax on
mammals, mice models may, for example, be used be used. Groups of outbred
ICR mice were injected intraperitoneally (i.p.) with Bacteriovorax and
bacteriophage cultures suspended in phosphate-buffered saline at various
PFU/mL. The interperitoneal route is generally the most sensitive in
determining virulence of microorganisms because the bacteria are injected
deep into the host into a site where they can multiply before an
effective host response can be mounted. We initially inoculated the
Bacteriovorax with a dose of about 1×109 PFU. The heath of
infected mice was monitored (i.e., death, scruffy fur, lethargy, or
change in rectal temperature) for one week. At the end of one week, mice
will be euthanized and the peritoneal cavity lavaged. The lavage fluid
will be examined for Bacteriovorax and phages by plaquing. Bacteriovorax
and bacteriophages in mixed suspensions were introduced orally into mice
to examine their potential replication and pathogenicity in the
intestinal tract. Mice will be starved of food and water for four hours.
Bacteriovorax-phage cultures will be suspended in PBS to a final
concentration of about 1010 PFU/mL. Mice will be fed 0.05 mL of 10
percent sodium bicarbonate to neutralize gastric acidity. Mice will then
be fed 0.02 mL of BALO-phage mixed suspension, via a micropipet tip
placed into the mouth. As above, the health of the mice will be monitored
for one week, at which time they will be euthanized. The intestines will
be removed, and the contents examined for HBALO by plaquing. A section of
intestines will be fixed in 10 percent formalin and examined for
histological damage by H&E staining. Samples of blood will also be
examined for plaquing activity.

[0110] Bacteriovorax and bacteriophages tested have not been shown to
possess the required molecular machinery to infect humans indicating the
safety of using the agents in combination to treat human bacterial
diseases. If using these biological agents to decontaminant harmful
bacteria before reaching human body such as foods for human consumption,
presumably, the higher dose may achieve quicker therapeutic effects. The
highest dose of Bacteriovorax and phages we have tested were about
4×108 PFU/mL, which were able to decrease by 5 logs of vibrio
vulnificus population within 40 hours in seawater. In embodiments, a
range of from about 1×106 to about 1×1010 for both
may, for example, be used.

[0111] When Bacteriovorax and bacteriophages were inoculated into cultures
of vibrio vulnificus, each was able to reduce the abundance of the prey
significantly at 40 hour as measure by plate count (FIG. 1). The killing
rate in microcosm Fl containing both Bacteriovorax and phage was
significantly higher than those with a single predator after 4 hours. At
40 hours, the effect of Bacteriovorax and bacteriophage in combination
was similar to that with Bacteriovorax alone, reducing the vibrio
vulnificus by approximately 4.4 logs, whereas, with phage alone it was
1.9 logs (FIG. 2).

[0112] FIG. 1 Kinetics of the lysis of prey cells over time in test with
Bacteriovorax (Bx)Bx and/or bacteriophages and control (with either
predators or no predators). Error Bars are standard error from three
independent experiments.

[0113] FIG. 2. Growth dynamics of Bacteriovorax and phage on prey over a
40 hour period as measured by plate counts. Broken lines designate phage
count; solid lines represent Bacteriovorax counts. F1 is the flask with
both Bacteriovorax and phage. F2 and F3 are the microcosms consisting of
vibrio vulnificus with Bacteriovorax and phage respectively.

[0114] Bacteriovorax was the first and greatest responder to the prey,
increasing 20-fold in plaque-forming units (PFU) to about
5×109 PFU ml-1 after about 12 hours incubation. The phage
response was weak, as evidenced by little increase in the F1 flask and
about a 5-fold increase in the F3 flask at about 40 hours. Both phage and
Bacteriovorax abundance decreased in the Fl flask caused by low number of
the remaining prey.

[0115] Results From Electron Microcopy

[0116] The results showed that Bacteriovorax and phage were able to infect
vibrio vulnificus cells separately (FIG. 3A & B) and jointly (FIG. 4).
Electron microscopy also confirmed that the Bacteriovorax, was effective
at infecting the prey, vibrio vulnificus. Micrographs suggest that
competition exists between Bacteriovorax and phages for the source of
food as they both are able to prey on the same bacterium. Here we report
a novel finding that Bacteriovorax and bacteriophages are able to infect
and reside in a single prey cell resulting from dual infection.

[0117] FIG. 3. TEM micrographs showing the predation of bacteriophages and
Bacteriovorax on prey vibrio vulnificus. The arrow in Figure A points to
a prey cell infected by phage CK2. The star indicates a cell infected by
Bacteriovorax to form a bdellobast (osmotically stable structure in which
the prey cell becomes rounded) and no phage is seen inside. Figure B
shows Bacteriovorax and phage both inside a vibrio vulnificus cell.
Additional evidence showing Bacteriovorax and phage are able to infect
the same cell.

[0118] While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or alterations of the invention. In general, the principles of the
invention and including such departures from the present disclosure as
come within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.

Patent applications in class Intentional mixture of two or more micro-organisms, cells, or viruses of different genera

Patent applications in all subclasses Intentional mixture of two or more micro-organisms, cells, or viruses of different genera